Single-cysteine substitution mutants at amino acid positions 306-321 in rhodopsin, the sequence between the cytoplasmic end of helix VII and the palmitoylation sites: Sulfhydryl reactivity and transducin activation reveal a tertiary structure

As sensors for structure at the cytoplasmic face of rhodopsin, single-cyste ine substitution mutants have been previously studied in the regions connec ting helices III and IV and helices V and VI. In this paper we report on si ngle-cysteine substitution mutants at amino acid positions 306-321, compris ing the cytoplasmic sequence between helix VII and the palmitoylation sites in rhodopsin. The cysteine opsin mutants were expressed in COS-1 cells and on treatment with Il-cis-retinal all formed the characteristic rhodopsin c hromophore. Cysteines at positions 306-316 and 319 reacted in the dark with the thiol-specific reagent 4,4'-dithiodipyridine (4-PDS) but showed a wide variation in reactivity. Cysteines at positions 317, 318, 320, and 321 sho wed no reaction with 4-PDS. The mutants on illumination also showed wide va riations in activating G(T). The mutant Y306C showed almost no G(T) activat ion, 1307C and N310C were poor, and the activity of the mutants M309C, F313 C, and M317C was also reduced relative to WT. The results suggest that the region comprising amino acids 306-321 is a part of a tertiary structure and that specific amino acids in this region on light-activation participate i n the interaction with G(T).